1. Field of the Invention
The present invention relates generally to the fields of gene therapy and biochemical pharmacology. More specifically, the present invention relates to mutants of human enzyme thymidylate synthase and uses thereof.
2. Description of the Related Art
Thymidylate synthase (TS, EC 2.1.1.45) catalyzes the rate limiting step in the sole de novo biosynthesis pathway to thymidylate, which is necessary for DNA synthesis and repair (Carreras et al., 1995). The mechanism of TS activity involves the reductive methylation of the substrate, 2xe2x80x2-deoxyuridine 5xe2x80x2-monophosphate (dUMP) by transfer of a methylene group from the cofactor, 5,10-methylenetetrahydrofolate (CH2H4folate), to generate 2xe2x80x2-deoxythymidine 5xe2x80x2-monophosphate (dTMP) and 7,8-dihydrofolate (H2folate). The inhibition of the TS pathway results in a thymineless state, which is toxic to rapidly dividing cells which have a high dTTP demand for DNA synthesis. This cytotoxicity is caused by DNA fragmentation and misincorporation of dUTP due to dTTP depletion. If there is enough supplied exogenous thymidine, cells survive through the salvage pathway depending on the use of thymidine kinase (TK). However, in normal tissues and in some tumor cells, the concentrations of circulating thymidine may not be sufficient to keep cells normally growing (Touroutoglou et al., 1996).
As a consequence, TS is an attractive target for anti-cancer drug design due to its crucial role in maintaining pools of thymidylate for DNA synthesis. Since the 1950s, many analogues of both the pyrimidine substrate (dUMP) and folate cofactor (CH2H4folate) have been synthesized and tested as potential anti-cancer therapeutics. However, although a number of inhibitors that tightly bind to TS were discovered, before 1995, 5-fluorouracil (5-FU) was the sole TS-targeted drug approved for clinical application. In vivo, 5-FU is metabolized to 5-fluoro-2-deoxyuridylate (FdUMP) that subsequently occupies the dUMP binding site forming a ternary complex with the enzyme and the folate cofactor, resulting in inhibition of TS. As the three-dimensional structures of TS have been revealed, the folate binding site in TS has been explored for the design of highly specific inhibitors (Jackman et al., 1995b), and have led to the emergence of novel folate analogues, such as tomudex (ZD1694), BW1843U89, AG331 and AG337 etc. These agents as the new generation TS-directed inhibitors have entered clinical trail in recent years. The approval of tomudex for treatment of advanced colorectal cancer in the United Kingdom occurred last year.
The major blood folate is 5-methyl-tetrahydrofolate (5-CH3xe2x80x94H4folate), which enters cells via membrane transports [or called reduced folate carriers (RFC)]. Once inside the cell, 5-CH3xe2x80x94H4folate is metabolized by methionine synthase to tetrahydrofolate. This coenzyme is converted by serine hydroxymethyltransferase to 5,10-methylene-tetrahydrofolate and also polyglutamated by folylpolyglutamate synthase (FPGS) to become 5,10-methylene-tetrahydrofolate polyglutamates [CH2H4folate(Glu)n]. CH2H4folate(Glu)n, as a cofactor, donates its one-carbon unit and two electrons to the reductive methylation reaction converting dUMP to dTMP. Dihydrofolate (H2folate) is a product of this process, which requires the sequential action of dihydrofolate reductase (DHFR) and serine hydroxymethyltransferase in order to resynthesize CH2H4folate(Glu)n. Inhibition of DHFR by methotrexate (MTX) may lead to an accumulation of folates in the inactive H2folate form, resulting in depletion of CH2H4folate and dTMP.
There is much interest in correlating enzyme structure and function using mutagenesis. To date, several hundred mutations have been made in L. casei, E. coli and human TS (Climie et al., 1990b; Michael et al., 1990). Most of mutations in L. casei were produced by cassette mutagenesis (Wells et al., 1985; Climie et al., 1990a). The synthetic L. casei TS gene was engineered by creating over 30 unique restriction sites about equally spaced throughout the entire gene, providing xe2x80x9creplacements setsxe2x80x9d in which several target amino acids were replaced by a large number of substitutions. Another approach involving the introduction of an amber stop codon were adopted to generate multiple mutants of E. coli TS (Michaels et al., 1990; Kim et al., 1992). Using these approaches the various mutants in either L. casei or E. coli system were first screened for catalytic activity of TS by genetic complementation in a TS-deficient E. coli host, and then mutants of interest characterized by kinetic studies. A few mutants of human TS and their expressed enzymes in mammalian cells have also been studied. The mutant human TSs were also tested to complementation of the growth of TS-negative E. coli stains in the absence of thymine to determine if the activity of an altered enzyme is sufficient to support growth. However, the correlation of a mutant human TS and drug resistance can not be interpreted by this complementation study in a bacterial system and mammalian cells lacking TS are required. The three-dimensional structure of human TS has provided the impetus to generate mutants of human TS having novel enzyme properties such as drug resistance.
Prediction of properties of enzymes obtained by site-directed mutations is poor. When the enzyme accommodates a single amino acid substitution, readjustment of neighboring residues may occur, resulting in structural plasticity. TS is one of best examples for observing this phenomenon. In general, TS can tolerate amino acid substitutions even in a highly conserved residue that is important for enzyme structure or function. In a few cases, a single amino acid replacement causing dramatic change in properties of TS was also found. By reviewing the mutations already made, it was found that highly conserved residues are hot spots for amino acid substitutions (Carreras et al., 1995), and there are a few residues such as Arg50, Glu87, Trp109, Cys195, Arg215, Asp218, and Tyr258 especially sensitive to substitution (Stroud et al., 1993). All of these residues are in the substrate or folate binding site.
Cys195 (ec146, lc198) involved in the binding of 2xe2x80x2-deoxyuridylate as well as initiating the catalytic process could only be modified to Ser for E. coli TS and still retain activity, albeit severely diminished activity. None of the comparable L. casei mutants showed detectable activity (Dev et al., 1988; Climie et al., 1990b). Conserved Arg residues at positions 50, 215, 175, and 176 form a positively charged binding surface for the phosphate anion of dUMP. In L. casei, For Arg175, another completely conserved residue could be replaced by a neutral (Ala, Thr), positive (Lys). or negative (Glu) amino acid without drastic changes in substrate binding or catalytic activity (Santi et al, 1990). Most substitutions for Arg176 of either E. coli or L. casei TS result in little impairment of function. In contrast, Arg218 could not resist any amino acid shifts.
The Arg50 loop, having less than 1.0 xc3x85 movement and reorientation upon Arg50 (ec21, lc23) binding to the phosphate of dUMP, is a highly conserved region. For Arg50, only four amino acids (Gly, Pro, Ser, and His) in E. coli TS and three residues (Val, Ile, and Gln) in L. casei TS are substitutable with retention of 10-50% of the wild-type activity (Zhang et al., 1990; Michaels et al., 1990). Asp49 (ec20, lc22) is quite sensitive to mutagenesis, except for replacements by the two polar (Cys, Ser) and one acidic (Glu) residues, all E. coli Asp49 mutants do not complement growth of TS-negative cells. In E. coli TS, Thr51 (ec22, lc24) tolerates substitutions of Pro, Ser, Tyr, Gln and Lys. Surprisingly, contrary to those neighbor residues, Gly52 (ec23, lc: His52) accepts any mutations. This residue has apparent reorientation upon the formation of ternary TS complex (Kim et al., 1992).
Trp109 (ec80, lc82) and Asn112 (ec: Trp83, lc: Trp85) are highly conserved residues that form hydrophobic contacts with both dUMP and CH2H4folate. Trp109 activity could not be fully restored by any of the substitutions except phenylalanine for E. coli TS, but showed high activity by three amino acid (Phe, Tyr and His) changes for L. casei TS (Michaels et al., 1990). Asn112 was only mutated to Phe for L. casei TS and the altered enzyme remained functional, but the W109F/N112F double mutant of L. casei was inactive (Carreras et al., 1995). Phe59 (ec30, lc32) forms part of the substrate binding pocket in tertiary structure. Leu and Tyr replacements for Phe59 of E. coli TS yield enzymes that complement the TS-deficient E. coli strain, but TS activity was totally lost for other substitutions (Kim et al., 1992).
The C-terminus region of TS plays a critical role in folate binding and catalysis (Perry et al., 1993). Deletion of just the residue Val313 results in TS protein that can bind both ligands but is catalytically inactive because the protein is incapable of closure to sequester the reactants. However, Val313 could tolerate almost all substitutions and many mutants were as active as wild-type TS (Climie et al., 1992; Carreras et al., 1992). A few mutagenesis studies for human TS have been published. Gln214, being believed in a kink region for three (xcex2-sheet formation of the central core of the polypeptide, is highly conserved in all TSs. Cell growth of the TS-negative E. coli was supported by Glu, His, Lys, or Ala, but not by Ser, Cys, or Trp substitutions (Zhao et al., 1995).
Until recently, only one mutation in TS has been reported to be related to TS-directed drug resistance. Tyr33 of human TS is one of 40 amino acid residues that are invariant among all reported TS sequences. The Tyr33 to His33 substitution was discovered in a human colon tumor cell line and conferred approximately a 3- to 4-fold resistance to FdUMP, a metabolite of the chemotherapeutic prodrug 5-fluorouracil. This mutation affects the catalytic properties of the TS enzyme, showing an 8-fold drop in kcat for the reaction. The Km values for both dUMP and CH2H4folate were not significantly different between the mutant and wild-type TS.
The crystal structure of human TS has shown that the side chain of Tyr is not directly involved in ligand binding site of the human TS. However, the hydroxyl oxygen of Tyr33 is hydrogen bonded to the backbone carbonyl oxygen of residue 219 at the first turn of the central hydrophobic helix J (residues 219-242). The first turn of helix J is consisted of eight amino acid residues (219-226), five of which are highly conserved and two (Leu221 and Phe225) form a hydrophobic pocket for the PABA ring of the cofactor. The drug-resistant mutation can be interpreted in terms of induced change by reorientation in the initial turn of the helix J to be no longer optimal for ligand binding. Why some substitutions are active in E. coli but not in L. casei TS, and vice versa is not known.
The discovery and development of TS inhibitors was based on molecular structures and properties of TS and its pyrimidine substrate or folate cofactor, especially as the three-dimensional structures of several unliganded and liganded TSs at the atom level of resolution were achieved. The first compounds to have clinically significant TS-inhibiting activity were the fluoropyrimidines 5-FU and FdUrd, which are metabolized to 5-fluorodeoxyuridine monophosphate (FdUMP) that subsequently occupies the substrate binding site leading to a stable and inactive TS complex. In addition, they also may be incorporated into RNA or DNA via fluoro-UTP or 5-fluoro-dUTP, respectively. Therefore, fluoropyrimidines are not pure TS inhibitors and are susceptible to metabolic degradation in vivo. In contrast, folate analogues may be designed as more specific and more stable TS-specific inhibitors. Moreover, the cofactor CH2H4folate is a relatively large molecule, which provides a variety of sites, amenable to manipulation in drug design (Schoichet et al., 1993).
ZD1694 and BW1843U89 are new, promising antifolates that are derived from the CB3717 chemical scaffold, which are characterized as classical antifolate TS inhibitors. They contain a glutamate moiety and can be metabolized to noneffluxable polyglutamate forms within the cell. The polyglutamylated TS inhibitors bind tighter than the corresponding monoglutamylated forms. By comparison, the nonclassical antifolate TS inhibitors. such as AG337 and AG331, lacking the glutamate, have recently been developed.
The antineoplastic agent 5-FU is a mechanism-based inhibitor of TS, which is metabolized to FdUMP that forms a stable covalent adduct with CH2H4folate as a steady-state intermediate, resulting in inhibition of TS. CB3717 (N10-propargyl-5, 8-dideazafolic acid), a lead compound as analogue of CH2H4folate, is a 2-amino-4-hydroxy quinazoline carrying a propargyl group on N-10 that greatly increases the affinity of TS, with Ki of 2.7 nM. CB3717 demonstrated antineoplastic activity in Phase I trials, but its development was abandoned due to unpredictable severe renal and hepatic toxicity caused by its poor aqueous solubility. Tomudex (N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methyl amino-]-2-thenoyl)-L-glutamic acid) was developed based on the molecular structure of CB3717. This quinazoline folate analogue, designed to be more water-soluble than CB3717 to avoid some side-effects such as nephrotoxicity, is a highly selective inhibitor of mammalian TS (Jodrell et al., 1991). Similar to CB3717, tomudex results in decreased TMP production, which leads to inhibition of DNA synthesis, resulting in cell death (Jackman et al., 1991a, b, and c).
Tomudex is a mixed noncompetitive TS inhibitor. In contrast to CB3717, tomudex enters cells using the reduced folate carrier (RFC). In the cell it is an excellent substrate for FPGS with an affinity 30 times higher than that of CB3717, and it is rapidly polyglutamylated by FPGS. The polyglutamated forms (n=2-6) are up to 100-fold more potent inhibitors of TS than is the monoglutamate. The polyglutamates are retained within cells, leading to a prolonged inhibitory action even in the absence of extracellular compound (Jackman et al., 1993). Tomudex thus is 500-fold more active in inhibiting cell growth than CB3717, despite being 20 times less potent as a TS inhibitor in enzyme assays (Ki, 60 nM) (Gibson et al., 1993; Lu et al., 1995). Tomudex has demonstrated activity in colorectal, breast, and pancreatic cancer and was approved in the U.K. for treatment of advanced colorectal cancer in August 1995. Also, phase III trials of tomudex in advanced colorectal cancer showed that tomudex is slightly superior to 5-FU with respect to anti-advanced colorectal cancer activity and therapeutic margin. BW1843U89 is an extremely potent, noncompetitive TS inhibitor in enzyme assays (Ki, 90 pM). As a TS-directed inhibitor, the monoglutamated form of BW1843U89 is as potent as the polyglutamated derivatives of tomudex in vitro studies. Similar to tomudex, growth inhibition could be reversed by thymidine alone, indicating that TS is its exclusive site of action. BW1843U89 does not require the RFC for the cellular entrance and is an excellent substrate for FPGS, but is only metabolized to a diglutamated form. The polyglutamation of this antifolate leads to retention in cells.
Drug resistance is a major obstacle to the successful use of chemotherapeutic agents in the treatment of neoplastic disease. For maintaining efficacious drug therapy, discovering new antitumor agents is an important goal. For drug resistance, investigations of naturally occurring resistance in model cell lines provides insights into the mechanisms that underlie innate clinical resistance in patients not previously exposed to these new drugs. The prior art is deficient in the lack of effective means of inhibiting the overcoming the resistance to TS inhibitors routinely encountered in anti-neoplastic therapy. The present invention fulfills this longstanding need and desire in the art.
The present invention randomly mutated HT1080 cells and subsequently selected drug-resistant clones with a high concentration of AG337. Secondly, site-directed mutagenesis was performed on three codons that code for amino acids that are folate-binding sites of human TS gene, based on the knowledge of three-dimension structures of TS. Using these two approaches, isolation and characterization of mutants of human TS conferring drug resistance to TS specific inhibitors were studied. The human TS mutants obtained have desirable properties including antifolate resistance, a high catalytic efficiency and good stability. This kind of TS variant is an excellent candidate for gene therapy approaches, namely to transfer drug resistance to human hemotopoietic progenitors, thus allowing dose-intense therapy in cancer patients by protecting normal cells and preventing dose-limiting myelotoxicity. Moreover, these mutants may be used as dominant selectable markers in therapeutic gene transfer protocol.
In one embodiment of the present invention, there is provided a mutated human thymidylate synthase, said mutated synthase differing from wild type thymidylate synthase of the amino acid sequence disclosed in Genbank Accession number NP001062 (SEQ ID No. 39) at amino acid residue 49, amino acid residue 52, amino acid residue 108, amino acid residue 221 or amino acid residue 225.
In another embodiment of the present invention, there is provided a cDNA encoding the mutated human TS of the present invention.
In yet another embodiment of the present invention, there is provided a DNA vector comprising: DNA encoding a mutated human TS of the present invention.
In still yet another embodiment of the present invention, there is provided a host cell transfected with the DNA vector of the present invention and wherein said host cell produces a mutated human TS.
In still yet another embodiment of the present invention, there is provided a method of decreasing the toxic effects of anti-neoplastic inhibitors of TS in an individual in need of such treatment, comprising the steps of: introducing a mutated human TS into cells of said individual; and returning said cells to said individual.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.